Project Summary Cell fate acquisition is a fundamental developmental process in all multicellular organisms and a growing body of evidence indicates that gene regulatory networks (GRNs) play an important role. However, the molecular regulation of the entire pathway from stem cell to differentiation has never been defined for any tissue. The Arabidopsis root, with its simple structure and defined stem cell niche, is a tractable model for studying the transcriptional regulation of cell fate. Over two decades of work have outlined the GRN that orchestrates cell proliferation and specification of the endodermis, a tissue analogous to the mammalian epithelium. This GRN is mapped in sufficient detail to now mathematically model its dynamics. However, crucial questions remain regarding downstream differentiation events. These include what regulators control endodermal fate stabilization and differentiation? And how closely are such regulators connected to the transcriptional events controlling stem cell proliferation? Until recently, technological constraints made it very difficult to study the molecular dynamics underlying development of a single cell type in the context of an entire organ or organism. The research proposed here utilizes new advances in imaging and transcriptional profiling to study protein and gene expression dynamics at cellular resolution. The overall goal of this proposal is to expand the topology of the endodermal GRN and begin to quantify the dynamics underlying differentiation. To achieve this goal, the proposed specific aims include conducting a forward genetic screen in a sensitized genetic background to uncover novel regulators of endodermal identity (Aim 1). In parallel, single cell RNA-sequencing experiments will define gene cascades underlying cell maturation events, thus providing systems-level insight into regulation of the entire pathway from stem cell to differentiated endodermis (Aim 2). To experimentally quantify the dynamics of known and novel regulators in the context of differentiation, state-of-the-art imaging techniques will be employed to track changes in protein accumulation over time at a cellular resolution in living roots (Aim 3). Together, these aims should expand the architecture of the endodermal GRN and begin to define how information flows through it to orchestrate cell differentiation events. The intellectually stimulating and collaborative environment of Duke University, coupled with individualized mentoring and enabling technology in the sponsoring lab, provide a resource-rich environment in which to conduct the proposed experiments. This research plan will facilitate advanced training in genetics, systems-level transcriptional regulation, multi-dimensional data analysis, and time-lapse microscopy, thereby providing the foundation for a long-term research program to dissect and model the GRNs underlying fundamental developmental processes. Insights gained from this work will deepen our mechanistic understanding of how stem cell progeny traverse the pathway to differentiation, thereby producing methodological and conceptual advances to inform tissue regeneration.